Project Summary/Abstract Our genomes experience a large amount of DNA damage, from both exogenous sources and endogenous byproducts of normal cellular processes. The ability of the cell to recognize and repair DNA damage is essential for maintenance of genomic integrity. Failures in DNA repair can lead to mutations, cell death, premature aging, and cancer. The work proposed here aims to analyze repair of highly deleterious type of DNA damage, a mitotic DNA double-strand break (DSB), and to determine the mechanism by which DSBs are repaired in the context of a multicellular organism, specifically Drosophila melanogaster. DSBs are repaired in different ways, including non-homologous end joining, where the ends are modified and joined, and homologous recombination (HR), where identical sequences are used to as a donor template to repair the DSB. Normal cells are highly sensitive to homology of the donor template, as recombination repair between diverged sequences is highly suppressed in single cell systems, thus avoiding genome rearrangements and instability. There are several mechanisms that determine which repair pathway is utilized, including sequence divergence, cell cycle, and genomic context of the DSB. While much of this work has been established in single-cell systems, the mechanisms that determine repair pathway choice have not been elucidated in multicellular organisms. To gain insight on DSB repair in the context of a multicellular organism, we previously used the novel DR-white DSB reporter assay to show that HR is the preferred DSB repair pathway in the male germline. However, it is unclear if HR is also the preferred pathway in other tissues with a canonical cell cycle (such as somatic tissues), or in tissues with non-canonical cell cycles (such as embryonic syncytial cycles). These questions will be addressed in Aim 1, utilizing DR-white genetic assay and molecular assays to determine which repair pathways are utilized in multicellular organisms. It is also unclear how mechanisms of end resection, which is required for HR, and homolog availability, a potential donor sequence for HR, drive repair pathway choice. To address this in Aim 2, we will use two novel genetic assays called Sce.white and iwhite2 to determine the kinetics of DNA end resection and measure interhomolog recombination and how perturbing this may impact repair pathway choice. Lastly, we previously used the novel DR-white.mu DSB reporter assay to show that the mismatch repair machinery modulates suppression of recombination between diverged sequences. However, additional genetic components responsible for this suppression and whether this phenomenon impacts repair pathway choice remain to be determined. These will be addressed in Aim 3, using reporter assays in Drosophila S2 cells. Importantly, to maintain goals of the AREA award mechanism, this project will give undergraduate researchers hands-on experience in a wide variety of molecular and genetic techniques and hypothesis-driven training, which will provide a valuable skill set for a future biomedical research and/or health-related careers.